DNA topoisomerase I and II (top1 and top2) are primary targets for cancer chemotherapy and can be inhibited by physiological and carcinogenic DNA modifications. Topoisomerase poisons act by stabilizing enzyme-linked DNA breaks which can be detected as protein- associated DNA breaks in drug-treated cells (cleavage complexes).We had previously reported that various DNA modifications including mismatches, abasic sites and DNA strand breaks were efficient top1 poisons. We now have extended these studies to DNA base modifications including oxidative base damage (8-oxoguanine & 5-hydroxycytosine) and carcinogenic adducts (ethenoadenine and benzo[a]pyrene guanine adducts), and demonstrated for the first time the induction of top1-DNA adducts in cells exposed to the carcinogenic isomer of benzo[a]pyrene diol epoxide. This observation suggests the possible involvement of top1 in the mutations observed after carcinogenic exposure. It also demonstrates how minor groove alkylation can trap top1, which is relevant for drug development as we found that the minor groove guanine N2 alkylator ecteinascidin 743 is a top1 poison.Because topoisomerases exist both in normal and cancer cells, the mechanism of drug selectivity for cancer cells are a focus of our studies. Our goal is to elucidate the antitumor mechanisms of topoisomerase poisons, and their selectivity for cancer cells. Secondary targets which are different in cancer cells could be used for drug development possibly in association with topoisomerase inhibitors or other DNA damaging agents.By studying the panels of human breast cell lines from the NCI Anticancer Drug Screen, we have investigated the relationship between apoptosis and cytotoxic response, and the parameters that are best correlated with cytotoxicity and could be used in the clinic to predict and monitor the response to topoisomerase inhibitors. Top1 protein levels appear comparable among cell lines and thus are not predictive, while top1 cleavage complexes are better correlated with cytotoxicity. Cleavage complexes are, however, not always predictive, and it appears that cellular response, including apoptosis and cell cycle checkpoints play an important role for drug response.We have found that mutagenic activation of the ras oncogene is associated with enhanced sensitivity to top2 inhibitors by a mechanism that involves enhanced apoptosis while cleavage complexes are not significantly modified. We have found that the DNA damage and stress activated gene Gadd45, also enhances the activity of topoisomerase inhibitors by destabilizing chromatin structure.The selective top1 inhibitors, camptothecins induce DNA damage in replicating cells by inducing the formation of DNA double- strand breaks. Cells generally respond to this effect by arresting in S-phase. The factors that mediate this replication in trans have been investigated. We found that DNA-dependent protein kinase (DNA-PK) is rapidly activated in response to camptothecin-induced replication- mediated DNA damage, probably as result of Ku binding to the ends of the DNA double-strand breaks after replication fork run-off. The middle subunit of replication protein A (RPA2) is rapidly phosphorylated by DNA-PK under these conditions, and remains phosphorylated until cells recover from S-phase and DNA synthesis arrest. These observations suggest that DNA-PK and RPA2 phosphorylation play a role in the S-phase checkpoint elicited by top1-mediated DNA damage. - chemotherapy, DNA adducts, DNA binding proteins, DNA repair, Pharmacology,